The tools of personalized medicine have revealed the powerful potential of biospecimens while also demonstrating their fragility, creating worry about how they’re collected and managed.

“I’m sitting in the cockpit of the war on cancer and I see it coming like a freight train,” says pathologist Carolyn Compton, MD, PhD, of the potential and the pitfalls.

As director of the Office of Biorepositories and Biospecimen Research (OBBR) of the National Cancer Institute, Dr. Compton comes to work each morning to see that some of the most valuable resources for modern medical research don’t end up like proverbial bugs on the windshield of that cockpit.

“Biospecimens are living, changing, biologic entities capable of reacting to their environments, and there is no environmental stress that is the biologic equal to the operating room or the pathology suite,” Dr. Compton says. She recites the many ways biospecimens are abused—industrial-strength drugs, anesthesia, dramatic changes in temperature, anoxia—that compromise integrity, utility, and shelf life.

In the brave new world of personalized medicine, she says, pathologists need to think about how specimen handling can change the molecular profile. “We don’t think about how long it sits at room temperature and what that means for desiccation rates,” Dr. Compton says. “But until we have absolutely suspended that tissue in animation—rendered it incapable of further biologic reaction—it keeps changing its molecular profile in reaction to what we are doing to it. We have to know what happened to it in the same way that we record what happens to the patient in the medical enterprise.”

“This will require a whole new thinking process,” says Dr. Compton, former chair of and now the NCI liaison to the CAP Cancer Committee.

The ability to derive genetic information from a patient’s cells and use that information to sculpt unique treatments will rely on airtight systems for collecting, studying, and storing biological materials. “The pathologist and the specimen will be the centers of the personalized medicine universe, because all of the molecular data that will guide patient care will in fact be derived from the patient’s own cells and tissue,” Dr. Compton says. “Technologies that are available right now or on the horizon will give us the ability to look at tens of thousands of RNA molecules or tens of thousands of proteins at one time, interpret them, and make the information medically meaningful.”

Molecular testing is more sensitive than light microscopy to fixation and storage methods, which is why Dr. Compton and her team at the OBBR are working to standardize biospecimen management—so that researchers at a variety of sites can work with tissues of uniform quality. The majority of the millions of specimens stored in tissue banks around the world, as it turns out, are not proving to be useful for newer research methods.

Biospecimen research depends on myriad variables. Temperature, number of minutes from excision to bench, time required for tissue to be fixed, method of storage, and materials employed all affect gene expression and the ability to detect protein biomarkers. Last year, recognizing that a protocol was needed to protect the integrity and govern the collection, preparation, and storage of cancer biospecimens, the NCI developed standards for frozen biospecimen management and published the “NCI Best Practices for Biospecimen Resources” (http://biospecimens.cancer.gov), which consist of principles by which lab procedures should be developed. Companion NCI tools for robust informatics, the NCI Biospecimen Research Database (http://brd.nci.nih.gov/BRN/) and the cancer Biomedical Informatics Grid (caBIG) (https://caBIG.nci.nih.gov), are also in place.

The OBBR is also project manager for The Cancer Genome Atlas (TCGA) project, a three-year pilot sponsored by the NCI and the National Human Genome Research Institute. Dr. Compton describes the genome atlas as a “big team science project” involving 10 large academic medical centers with genomics research power and expertise. The atlas pilot targets three cancers: glioblastoma multiforme, squamous cell carcinoma of the lung, and serous cystadenocarcinoma of the ovary. TCGA researchers, Dr. Compton says, “are working together to use every major genome analysis technology that we’ve got right now to interrogate the genome of three cancers to determine if a coordinated, systematic, standardized effort to do this in a team will yield new discoveries faster.”

The genome atlas is a public service project much like the human genome project, Dr. Compton says; all data will enter the public domain to create a new dictionary of genomic changes in cancer available to investigators worldwide. But despite an intensive international search, acquiring tissue for the pilot has been a challenge. “It’s really created a new awareness that we do not have the biospecimen collections we need to efficiently take full advantage of the scientific and technological power we now have to focus on cancer,” Dr. Compton says.

The demands of high-throughput research will call for large numbers of samples, and the samples in every institution have to be comparable if researchers are to derive harmonized results. The lack of high-quality, properly annotated biospecimens is, in Dr. Compton’s words, “a roadblock to translational research.”

“The attrition rate for qualification for [the genome atlas] is enormous,” Dr. Compton says, “and once the RNA and DNA have been extracted, there’s another drop based on the lack of quality in the biomolecules they don’t meet the quality control parameters for the RNA and DNA. It’s such a struggle, because people weren’t aware that they didn’t have what they thought they had. In the biobanking world, you put stuff into the bank but you don’t really know what you’ve got until you pull it out of the bank because that’s when the quality control takes place.” They have had to put institutions under contract to collect specimens prospectively from new patients, using the provided protocols that will yield the types and quantity of specimens needed. “It was a wake-up call,” Dr. Compton says.

Pathologists already have the skills and training they will need to manage biorepositories, she says. “It’s just a matter of using the expertise they reserve for patient care with an eye toward research and preserving the tissue for research purposes,” she says. “So selecting areas of the tumor that are not necrotic, that are not hemorrhagic, that are tumor rich, for which you have validated the diagnosis. And it all has to be annotated, which requires time and expertise.”

Cancer advocacy groups like the Susan B. Komen and Lance Armstrong foundations are already up on biorepository science. “Advocacy groups realize that biospecimens are a major issue,” Dr. Compton says. “There’s barely a one that doesn’t recognize the central importance of high-quality biospecimens in making the science go forward.” The involvement of patient groups makes it clear that physician advocacy for billing codes to allow reimbursement for biospecimen management is not self-serving. “Pathologists already have too little time to address the issues that they’re professionally responsible for. To add this to their workload without any compensation seems unreasonable,” Dr. Compton says. “Advocates recognize that this needs to occur, and they can be our voice on a congressional level to make it happen.”

Another NCI pilot, the National Community Cancer Centers Program, is bringing cutting-edge tools to communities distant from an NCI-designated Comprehensive Cancer Center. “Eighty percent of cancer patients get taken care of in the community, not in NCI-designated cancer centers,” Dr. Compton says. Through the NCCCP, “we can take the science to where the patients are.”

In his message on the NCCCP Web site (ncccp.cancer.gov), NCI director John E. Niederhuber, MD, a surgical oncologist, talks about the hope that this three-year pilot will launch systemic changes leading to earlier detection, better outreach in medically underserved communities, and more consistent followup. He points out that when community hospitals adopt standards for biospecimen collection and storage along with the tools to access and contribute to a sophisticated shared database, private-practice oncologists and their patients will have access to translational research.

The 16 pilot sites in 14 states that were selected to launch the NCCCP pilot last year are community-hospital–based cancer centers, under the direction of a physician with cancer expertise, that offer multispecialty care, see at least 1,000 new cancer cases annually, participate in clinical trials, and take a leadership role in health care outreach for underrepresented and disadvantaged populations. The pilot has four goals: give more patients access to clinical trials; reduce the disproportionate burden of cancer among underserved populations; give more patients the opportunity to provide biospecimens while assessing how the NCI best practices guidelines can be applied nationwide; and study the viability of a national electronic medical record database.

James A. Robb, MD, is a contracted consulting pathologist to the NCI and the OBBR who works with Dr. Compton to coordinate the biospecimen portion of the NCCCP. The NCI best practices that are published are for frozen tissue, he says; the focus now is on completing best practices for formalin-fixed, paraffin-embedded tissue. The hope, he says, is that draft guidelines can be completed within about a year.

“It’s very difficult for community hospitals to handle the frozen tissue,” says Dr. Robb, a member of the CAP Board of Governors. “They don’t have the liquid nitrogen system or the staff to carry it back and forth. We’re going to need robust, clinically valid molecular assays in formalin-fixed, paraffin-embedded tissue, but to do that we have to have a uniform way of fixing it.” OBBR would like to see it snap-frozen in liquid nitrogen and formalin-fixed within 20 minutes of removal from the patient so specimens would be of uniform quality. “We want a piece of the viable cancer within 20 minutes of when it comes out of the patient. Community hospitals without snap-freezing capability should cut the viable cancer tissue in 2- to 3-mm thick sections and place it directly into neutral-buffered formalin. If that can be accomplished, we will have uniform, high-quality paraffin blocks for both research and patient testing.”

To develop the best practices for a formalin-fixed, paraffin-embedded repository, Dr. Robb says, baseline quality control molecular markers will have to be identified for those blocks. “We want to do that so pathologists can go back into their paraffin-block storage facility, where many years of blocks are stored, take those blocks, test them for the quality markers, and get an ‘A’ quality block, ‘B’ quality block, ‘C’ quality block, and so forth. This quality testing will determine which blocks can be used for each type of molecular testing.”

The effect of tissue fixation methods on molecular stability was not understood (or relevant) before molecular testing. “Now we have to understand and deal with all the stress-related molecular states that change biomarkers and regulatory pathways other than DNA, which is stable,” Dr. Robb says. “RNA and proteins are changing rapidly after the blood supply to the cancer is interrupted and when the biospecimen is moved from 37° to 20°F, causing a significant temperature change. We’ll never be able to test the biospecimen in real time in vivo because of these changes, but if we can get the biospecimen out within 20 minutes and get it frozen or formalin-fixed in small 5-mm3 frozen and 2- to 3-mm thick formalin pieces, we’ll have uniform specimens. That’s what developing quality biospecimens is all about. In addition, we will have to produce high-quality clinical pathology specimens.”

For the National Community Cancer Centers Program, the NCI has created a 112-item checklist based on the best practices and asked the 16 community hospitals in the three-year pilot to figure out how their current cancer care practices compare with the ideal model, then speculate how they might close gaps if they had the resources.

The community cancer centers are not in competition with the established comprehensive cancer centers, he explains. “They all have to have relationships with academic centers so they can send complicated cases to them,” he says. The sites are in areas where there are no academic centers and where there are significant problems of access to cancer care. Some of the sites are setting up their own biorepositories, while others will continue to send biospecimens to academic centers.

Patients at NCCCP sites have access to clinical trials. “Only about four percent of U.S. patients who qualify for NCI-sponsored cancer clinical trials are participating,” Dr. Robb says, “and this low participation rate produces a major block for getting research biospecimens.” This problem can potentially be overcome, he says, through the use of the developing initiatives in molecular testing, bioinformatics with multidirectional IT connectivity such as caBIG, high-quality biospecimens, and “what is really essential—attaching the appropriate and complete clinical information to the biospecimens.”

Researchers have learned, he says, that molecular test results are affected by the patient’s diagnosis, type and length of anesthesia, amount of blood loss, blood pressure, drug therapy, and other clinical factors. Detailed clinical annotation is key. “As long as you can document it, you can go back and look at these specimens and see what was clinically relevant,” Dr. Robb says. “If the specimens are really high quality and collected uniformly, we’ll be able to translate the research to patient care much more quickly. That’s the whole translational piece: from bench to bedside to bench.”

Key to this personalized health care effort, he says, will be the pathologist participating effectively in the patient treatment teams—being a clinician.

The procedure to get high-quality formalin-fixed, paraffin-embedded tissue is no different from what pathologists are doing now, Dr. Robb says. “They need to get it into formalin in 2- to 3-mm thick pieces within 20 minutes of out-of-body, and they need to fix it in formalin for at least six hours but no more than 48 to 72 hours, including processing time. The upper limit for time in formalin is still being studied and discussed but should be no more than 72 hours.” He notes that the approximate time in formalin should be documented in the final pathology report. “A simple default comment could be:‘This specimen was in formalin between six and 72 hours.’ If the time in formalin was not within this range, the approximate time should be specifically provided.”

Pending the success of the ­NCCCP pilot in two years, the NCI will expand it to more facilities. “We’ll know how to do it better,” Dr. Robb says. “NCI will take its best practices and expand the project, so, hopefully, within a decade the majority of community cancer centers throughout the U.S. will have these capabilities.”

CAP governor Jay F. Schamberg, MD, chaired a CAP ad hoc steering committee formed two years ago to consider a proposal from Dr. Compton for a collaboration under which the CAP would help the NCI develop protocols for biospecimen management. “The concern at NCI was that they had no way of assuring themselves that the biorepositories they fund as a federal agency are of the highest and consistent quality,” Dr. Schamberg says.

That discussion culminated in a preliminary pilot under which three Biospecimen Best Practice Protocols developed by the NCI are being reviewed by resource committees of the CAP Council on Scientific Affairs. The CAP Standards Committee will coordinate their work. The three protocols address tissue formalin-fixed, HER2 (ERBB2) biospecimens to be examined via immunohistochemistry; tissue formalin-fixed HER2/ FISH/ SISH/ CISH to be examined via fluorescent in situ hybridization (FISH); and serum/plasma prostate specific antigen assay.

Paul N. Valenstein, MD, vice chair of the Council on Scientific Affairs, says the College is approach­ing the proposed full-blown collaboration with caution because the project, while worthwhile, is potentially monumental. “There are hundreds of assays for which best practices can be written, and biorepositories also need to think about how to store specimens for assays that have yet to be developed.” The NCI is interested in biospecimen best practices for cancer research, Dr. Valenstein acknowledges. But, he says, biorepository directors in patient care facilities also need to consider how best to store specimens for the benefit of patients who contributed them—patients who may someday need testing to guide future therapies. “Is it practical to ask hospital laboratories to store frozen specimens of tumors for tests that do not yet exist?” Dr. Valenstein asks. And, he notes, biorepositories that support transplantation and forensic testing face unique challenges.

The NCI does not seem to be interested in regulating biorepositories, Dr. Valenstein says, so much as in developing a set of best practices that will help researchers make sure they have appropriate biospecimens for the types of assays they’re doing today. Still, the number of assays and tests could increase significantly as the science moves forward. The sheer volume of unknowns makes it impossible to determine the level of commitment that might be required of the College, let alone the volunteer time needed to develop and review protocols. So the decision was to do a pilot test to see how much work was involved in reviewing protocols for storing three types of biospecimens and for three types of assays. When that is done, the CAP will decide whether to continue this activity beyond the pilot stage.